Parvoviruses are unique among all known organisms in having DNA genomes which are both single-stranded and linear. Both 5'and 3'ends of the genome are palindromic and can fold into hairpin structures that give rise to the two viral replication origins in replicative-form (RF) DNA. In the homotelomeric parvoviruses these hairpins are part of a terminal repeat, whereas for the heterotelomeric subgroup, the termini are typically unrelated to one another in either sequence or structure. The long term goal of this research program is to understand, at the molecular level, how heterotelomeric parvoviruses inveigle their host cell into replicating and packaging such apparently alien molecules. The major model we will continue to explore is the autonomous parvovirus Minute Virus of Mice (MVM), a genetically tractable virus that grows in cell culture. We will use what we learn from MVM to explore the function of the initiator protein of the newly-discovered pathogenic human bocavirus HBoV, for which there is currently no cell culture system. We propose to build on our previous research on MVM DNA replication initiation to study the structure and function of the terminal hairpins, extending this to the precise sequence requirements for NS1 binding, melting and nicking at MVM OriL. We will explore the functional significance of NS1 binding sites embedded in the MVM genome, and ask whether a similar situation pertains for HBoV. We will initiate studies on how the virus activates cellular DNA damage responses and whether the unique pseudochromatin that the virus elaborates is part of its strategy to evade, or employ, these elements of innate host defense. Genetic and biochemical approaches will be pursued to identify the molecular motor that drives genome packaging, and to explore the function(s) of the teminal hairpins in viral DNA replication and packaging. PUBLIC HEALTH RELEVANCE: This research program aims to develop an understanding of the mechanisms underlying parvoviral DNA replication and packaging, and further our knowledge of parvoviruses as pathogens. These are also necessary prerequisites for the use of these viruses as vaccine vectors against infectious diseases and cancer. The newly discovered human parvovirus, HBoV, appears to be one of the most prevalent infections of childhood, and understanding its natural history and mode of replication will become of increasing importance as we discover more about its interaction with the human population. In the present renewal application, therefore, we propose to parlay what we know about parvoviral genome structure and DNA replication from the murine system into a study of similar processes that regulate replication of the human virus.